Aránzazu Valverde

Extended-spectrum β-lactamases (ESBLs) represent a major threat among resistant bacterial isolates. The first types described were derivatives of the TEM-1, TEM-2 and SHV-1 enzymes during the 1980s in Europe, mainly in Klebsiella pneumoniae associated with nosocomial outbreaks. Nowadays, they are mostly found among Escherichia coli isolates in community-acquired infections, with an increasing occurrence of CTX-M enzymes. The prevalence of ESBLs in Europe is higher than in the USA but lower than in Asia and South America. However, important differences among European countries have been observed. Spread of mobile genetic elements, mainly epidemic plasmids, and the dispersion of specific clones have been responsible for the increase in ESBL-producing isolates, such as those with TEM-4, TEM-24, TEM-52, SHV-12, CTX-M-9, CTX-M-14, CTX-M-3, CTX-M-15 and CTX-M-32 enzymes.

The occurrence of extended-spectrum beta-lactamase (ESBL)-producing isolates has increased worldwide. Fecal carriage of ESBL-producing isolates has mainly been detected in nosocomial outbreaks, and few studies have evaluated fecal carriage during nonoutbreak situations and among patients in the community. We have studied the prevalence of ESBLs in 1,239 fecal samples from 849 patients (64.1% of whom were ambulatory) in 1991 and have compared the prevalence data with those obtained in 2003 for 400 fecal samples from 386 patients (75.9% of whom were ambulatory) and 108 samples from independent healthy volunteers. Samples were diluted in saline and cultured in two MacConkey agar plates supplemented with ceftazidime (1 microg/ml) and cefotaxime (1 microg/ml), respectively. Colonies were screened (by the double-disk synergy test) for ESBL production. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis with XbaI digestion; and the ESBLs of all ESBL-producing isolates were characterized by isoelectric focusing, PCR, and sequencing. The rates of fecal carriage of ESBL-producing isolates increased significantly (P < 0.001) in both hospitalized patients and outpatients, from 0.3 and 0.7%, respectively, in 1991, to 11.8 and 5.5%, respectively, in 2003. The rate of occurrence of ESBL-producing isolates among healthy volunteers was 3.7%. All ESBL-producing isolates recovered in 2003 were nonepidemic clones of Escherichia coli. ESBL characterization revealed an increasing diversity of ESBL types: TEM-4 and CTX-M-10 were the only enzymes detected in 1991, whereas TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-10, CTX-M-14, and a CTX-M-2-like enzyme were recovered in 2003. The ESBL-producing isolates recovered from outpatients in 2003 corresponded to a CTX-M-9-type cluster (62.5%) and SHV-12 (31.2%), whereas TEM-4 was detected only in hospitalized patients. The frequencies of coresistance in isolates recovered in 2003 were as follows: sulfonamide, 75%; tetracycline, 64.3%; streptomycin, 57.1%; quinolones, 53.5%; and trimethoprim, 50%. The increased prevalence of fecal carriage of ESBL-producing isolates during nonoutbreak situations in hospitalized patients and the establishment of these isolates in the community with coresistance to non-beta-lactam antibiotics, including quinolones, represent an opportunity for these isolates to become endemic.

Fecal carriage of extended-spectrum-beta-lactamase (ESBL)-producing organisms was detected in 70% of index cases of patients (n = 40) with community-acquired infections due to ESBL producers and reached 16.7% in household contacts (n = 54). A total of 66% of ESBL-producing organisms from index cases were indistinguishable from isolates from household contacts by pulsed-field gel electrophoresis. Patients with community infections and members of their households represent a reservoir for ESBL producers, increasing dispersal of resistance in healthy people.

ABSTRACT The spread of extended-spectrum-β-lactamase (ESBL)-producing organisms, particularly those harboring the CTX-M-type enzymes, both in the hospital and in the community, is difficult to discontinue due to the successful mobilization and evolution of the genetic elements harboring ESBL genes and coresistance rates in these isolates. The activities of tigecycline against 285 non-clonally related isolates (172 from Escherichia coli , 84 from Klebsiella spp., 20 from Enterobacter spp., 5 from Salmonella spp., and 4 from Citrobacter spp.) expressing well-characterized ESBLs and recovered in our hospital and its community area of influence were comparatively assessed (CLSI microdilution). Susceptibility rates for meropenem, imipenem, tigecycline, amikacin, and piperacillin-tazobactam were 100%, 100%, 97.5%, 93.3%, and 93%, respectively. Tigecycline (mode MIC, 0.5 μg/ml; MIC 90 , 1 μg/ml) was 4- to 256-fold more active than doxycycline and minocycline (mode MIC range, 2 to 128 μg/ml). CTX-Ms were the most frequent ESBLs (61.4%), 65.8% in community and 58.6% in nosocomial isolates. CTX-M-9 (22%), CTX-M-14 (15.8%), and CTX-M-10 (14%) were the most represented derivatives. SHV and TEM variants constituted 22.8% and 15.8% of the ESBLs, respectively. Overall coresistance rates were as follows: gentamicin, 27.4%; tobramycin, 27.4%; amikacin, 6.7%; and chloramphenicol, 29.1%. Sulfonamide (61.7%), trimethoprim (52.3%), streptomycin (50.5%), and ciprofloxacin (37.2%) resistance levels were significantly ( P &lt; 0.001) associated with CTX-M-9 producers. No tigecycline resistance was observed, although seven Klebsiella pneumoniae isolates exhibited intermediate MICs (4 μg/ml). Tigecycline, lacking cross-resistance with other compounds, could represent an opportunity to reduce the intensity of selection for ESBL-producing organisms derived from the use of other antimicrobial agents. However, this in vitro promise requires support from clinical studies.

Since its first description in 2000, CTX-M-14 has become one of the most widespread extended-spectrum beta-lactamases in Spain. In the present Escherichia coli multilevel population genetic study involving the characterization of phylogroups, clones, plasmids, and genetic platforms, 61 isolates from 16 hospitalized patients and 40 outpatients and healthy volunteers recovered from 2000 to 2005 were analyzed. Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] type, phylogenetic group, multilocus sequence type [MLST]) was established by standard methods. Analysis of transferred plasmids (I-CeuI; S1 nuclease; restriction fragment length polymorphism analysis; and analysis of RNA interference, replicase, and relaxase) was performed by PCR, sequencing, and hybridization. The genetic environment of bla(CTX-M-14) was characterized by PCR on the basis of known associated structures (ISEcp1, IS903, ISCR1). The isolates were mainly recovered from patients in the community (73.8%; 45/61) with urinary tract infections (62.2%; 28/45). They were clonally unrelated by PFGE and corresponded to phylogenetic groups A (36.1%), D (34.4%), and B1 (29.5%). MLST revealed a high degree of sequence type (ST) diversity among phylogroup D isolates and the overrepresentation of the ST10 complex among phylogroup A isolates and ST359/ST155 among phylogroup B1 isolates. Two variants of bla(CTX-M-14) previously designated bla(CTX-M-14a) (n = 59/61) and bla(CTX-M-14b) (n = 2/61) were detected. bla(CTX-M-14a) was associated with either ISEcp1 within IncK plasmids (n = 27), ISCR1 linked to an IncHI2 plasmid (n = 1), or ISCR1 linked to IncI-like plasmids (n = 3). The bla(CTX-M-14b) identified was associated with an ISCR1 element located in an IncHI2 plasmid (n = 1) or with ISEcp1 located in IncK (n = 1). The CTX-M-14-producing E. coli isolates in our geographic area are frequent causes of community-acquired urinary tract infections. The increase in the incidence of such isolates is mostly due to the dissemination of IncK plasmids among E. coli isolates of phylogroups A, B1, and D.

ABSTRACT This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing bla CTX-M-9 , and its association with Tn 402 , Tn 21 , and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn 402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 ( n = 26), IncP-1α ( n = 10), IncFI ( n = 4), and IncI ( n = 1) groups. Genetic platforms containing bla CTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn 402 derivatives. They were associated with Tn 21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified bla CTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn 402 derivatives, often to Tn 21 , highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of bla CTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae .

Abstract We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 p m and a limit of detection of 1.2 p m for the methylated synthetic sequence of the tumor suppressor gene O ‐6‐methylguanine‐DNA methyltransferase ( MGMT ) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.

This paper reports the preparation of versatile electrochemical biosensing platforms for the simple, rapid, and PCR-independent detection of the most frequent DNA methylation marks (5-methylcytosine, 5-mC, and/or 5-hydroxymethylcytosine, 5-hmC) both at global and gene-specific levels. The implemented strategies, relying on the smart coupling of immuno-magnetic beads (MBs), specific DNA probes and amperometric detection at screen-printed carbon electrodes (SPCEs), provided sensitive and selective determination of the target methylated DNAs in less than 90 min with a great reproducibility and demonstrated feasibility for the simultaneous detection of the same or different cytosine epimarks both at global level and in different loci of the same gene or in different genes. The bioplatforms were applied to determine global methylation events in paraffin-embedded colorectal tissues and specific methylation at promoters of tumor suppressor genes in genomic DNA extracted from cancer cells and paraffin-embedded colorectal tissues, and in serum without previous DNA extraction from cancer patients.

In this work, the development of the first integrated electrochemical immunosensor for the determination of the IL-13Rα2 is reported. The strategy involves the immobilization of a biotinylated capture antibody onto streptavidin-modified screen-printed electrodes through grafting with p-aminobezoic acid (p-ABA) and further activation using EDC/Sulfo-NHS chemistry. A hybrid nanomaterial composed of multiwalled carbon nanotubes (MWCNTs) and Graphene Quantum Dots (GQDs) was used as nanocarrier of multiple detector antibody and HRP molecules. Amperometric detection with the system H2O2/hydroquinone (HQ) achieved a linear calibration plot ranging from 2.7 to 100 ng mL−1 IL-13sRα2, with a LOD value of 0.8 ng mL−1. The immunosensor showed an excellent selectivity and was successfully applied to the determination of the target receptor directly in small amounts of raw cellular lysates and extracts of paraffin-embedded tissues from patients diagnosed with colorectal cancer at different stages.

ObjectivesTo analyse the ongoing epidemiology of KPC-producing Enterobacteriaceae after a non-ST258 KPC-3-producing Klebsiella pneumoniae outbreak in a university hospital in Madrid, Spain.

Untreated wastewater, particularly from hospitals and other healthcare facilities, is considered to be a reservoir for multidrug-resistant bacteria. However, its role in the spread of antibiotic resistances in the human population remains poorly investigated. We used whole genome sequencing to analyze 25 KPC-2-producing Enterobacteriaceae isolates from sewage water collected during a 3-year period and three clinical Citrobacter freundii isolates from a tertiary hospital in the same collection area in Spain. We detected a common, recently described, IncP-6 plasmid carrying the gene blaKPC-2 in 21 isolates from both sources. The plasmid was present in diverse environmental bacterial species of opportunistic pathogens such as C. freundii, Enterobacter cloacae, Klebsiella oxytoca, and Raoultella ornithinolytica. The 40,186 bp IncP-6 plasmid encoded 52 coding sequences and was composed of three uniquely combined regions that were derived from other plasmids recently reported in different countries of South America. The region harboring the carbapenem resistance gene (14 kb) contained a Tn3 transposon disrupted by an ISApu-flanked element and the core sequence composed by ISKpn6/blaKPC-2/ΔblaTEM-1/ISKpn27. We document here the presence of a novel promiscuous blaKPC-2 plasmid circulating in environmental bacteria in wastewater and human populations.

To the Editor: A new methicillin resistance mechanism gene, a divergent mecA homologue named mecC (formerly mecALGA251), was recently described in Staphylococcus aureus (1). Methicillin-resistant S. aureus (MRSA) isolates carrying mecC have been recovered from humans, ruminants, pets, and other animals such as rats, seals, and guinea pigs (1–3). It has been suggested that mecC-carrying MRSA isolates might not be detected by using MRSA selective media (4). For mecC-carrying S. aureus isolates, cefoxitin MICs of 4–64 mg/L have been demonstrated (1–2,4), values that would normally include susceptible isolates, according to the epidemiologic cutoff value established by the European Committee on Antibiotic Susceptibility Testing (EUCAST; www.eucast.org). mecC-carrying S. aureus isolates have been classified as heteroresistant (5), and MICs can be affected by the drug-susceptibility testing method used (1,5). These observations led us to retrospectively investigate the presence of mecC gene in a set of 361 mecA-negative S. aureus isolates collected during 2009–2012 (Table), independently of their susceptibility to cefoxitin. Isolates were recovered from healthy carriers in livestock (n = 39), from wild animals (n = 254), and from wastewater (effluents) from an urban sewage plant (n = 68). Specific amplification of the mecC gene was performed as described (6). The mecC-carrying S. aureus isolates were tested by broth microdilution using Microtiter EUST plates (Trek Diagnostic Systems, East Grinstead, UK) for susceptibility to benzylpenicillin, cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, florfenicol, fusidic acid, gentamicin, kanamycin, linezolid, mupirocin, rifampin, sulfamethoxazole, streptomycin, quinupristin-dalfopristin, tetracycline, thiamulin, trimethoprim, and vancomycin. Additionally, susceptibility to oxacillin was determined by using microScan Gram Positive Combo panel 37 (Siemens, Erlangen, Germany). MICs were interpreted according to EUCAST epidemiologic cutoff values. Table Testing of Staphylococcus aureus isolates for presence of methicillin resistance mechanism gene mecC, Spain* mecC was detected in a total of 4 isolates from wild boar (n = 1), fallow deer (n = 2), and urban wastewater (n = 1); these isolates represent 1% of the 361 tested isolates. The 3 isolates recovered from animals were susceptible to all antimicrobial drugs tested other than β-lactams and to oxacillin (MICs 0.5–1 mg/L) but were resistant to penicillin (MICs 0.5–2 mg/L). Two of the isolates were resistant to cefoxitin (MICs 8 and 16 mg/L) and the third was susceptible (MIC 4 mg/L). The wastewater isolate was resistant to penicillin (MIC 2 mg/L) and erythromycin (MIC 16 mg/L) and susceptible to all other antimicrobial drugs tested, including cefoxitin (MIC 4 mg/L) and oxacillin (MIC ≤0.25 mg/L). Previous studies have described mecC-positive isolates as susceptible to all antimicrobial drugs tested except β-lactams (2,3), although sporadic resistance to fluoroquinolones has been found (4,7). We additionally found erythromycin resistance in 1 mecC-carrying S. aureus isolate. For the 4 mecC-carrying S. aureus isolates we detected, MICs of oxacillin were interpreted as susceptible, and 2 isolates were susceptible to cefoxitin according to EUCAST guidelines, findings that agree with previous reports (1–2,4). Thus, mecC presence is not always linked to resistance phenotypes for cefoxitin or oxacillin; such unclear findings could hinder the detection of mecC-carrying isolates. We further characterized the 4 mecC-carrying S. aureus isolates by spa typing and detection of Panton-Valentin leukocidin (PVL) toxin genes (6,8). Multilocus sequence typing (MLST) was performed according to Enright et al. (9) by using self-designed primers arc (down 5′-CGATTTGTTGTTGATTAGGTTC-3′), tpi (up 5′-CATTAGCAGATTTAGGCGTTA-3′), and yqiL (down 5′-GATTGGYTCACCTTTRCGTTG-3′). All 4 isolates were PVL negative. The 3 animal isolates were assigned to a new spa type (t11212) and to clonal complex (CC) 425 and sequence type (ST) 425 (Table). ST425 has been previously associated with mecC-carrying S. aureus isolates in cattle and humans (1–2); the animals we sampled were from a game estate and may have had contact with cattle and with urban wastewater. The wastewater isolate was assigned to spa type t843 and to a new allelic profile, ST2676, in CC130 (Table). ST2676 represents a single-locus variant of ST130 carrying a different allele for the gene aroE. MRSA isolates of CC130 have been associated with humans and animals (1–4,6). This result indicates that mecC-carrying S. aureus isolates can be found in urban wastewater, which may act as an environmental reservoir, as has been demonstrated for mecA-carrying S. aureus (10). In conclusion, we detected the methicillin resistance mechanism gene mecC in nonclinical S. aureus isolates from animals and urban wastewater in Spain. Although our data indicate that the frequency of this resistance mechanism is low, this gene appears to be expanding to new areas. Prospective studies should be performed to evaluate epidemiologic changes and to analyze the genetic lineages that carry this resistance mechanism.

Xenobiotic estrogens in the environment or diet have received much attention as a possible source of certain hormonal disease states in human and wildlife. Therefore, the detection of estrogenic activity of any substance, especially those related to the food industry, is important. The estrogenic activity ofp-hydroxybenzoic acid (PHBA), a compound related to a commonly used group of preservatives in food, cosmetic, and pharmaceutical preparations, was evaluated with immature and adult ovariectomized female mice (CD1) using two well-known bioassays. Subcutaneous administrations (sc) of different doses of PHBA were compared with estradiol (E2), and their effects on vaginal cornification and uterotrophic activities were evaluated. Different groups of animals were treated sc daily for 3 days with vehicle (corn oil, 0.3 ml/100 g), E2(1 μg/100 g), and PHBA (0.5, 5, 50, and 500 μg/100 g). Four days after treatment, PHBA produced a dose-dependent response on vaginal cornification and uterotrophic activity in both immature and adult ovariectomized mice. The relative uterotrophic potency of PHBA (500 μg/100 g) to E2(1 μg/100 g) was 0.0011 in immature mice and 0.0018 in ovariectomized animals.

ABSTRACT CTX-M-10 has been widely disseminated among multiple clones of several species of Enterobacteriaceae , harboring seemingly different plasmids, for over a decade in Ramón y Cajal University Hospital, Madrid, Spain. Cloning and sequencing of a 12.2-kb DNA fragment from plasmid pRYCE21 from Klebsiella pneumoniae strain KP4aC revealed a novel phage-related element immediately upstream of bla CTX-M-10 conserved among different CTX-M-10-producing strains. This is the first report showing an extended-spectrum-β-lactamase gene linked to a phage-related element.

To analyse all extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates recovered from 2001 to 2004 in our institution and to compare this period with that of 1989 to 2000.All K. pneumoniae isolates recovered during the studied period were screened for ESBL production. One isolate per patient was selected for ESBL characterization and for population structure, including phylogenetic groups, and plasmid analysis.Ninety-three (3.2% mean) ESBL-producing K. pneumoniae isolates recovered from 61 patients (26%, medical wards; 18%, surgical wards; 25%, ICU; and 31%, outpatients) were identified. Outpatients significantly increased (P < 0.01) when compared with 1989-2000 (7%). The number of different ESBLs increased with persistence of previously identified enzymes (TEM-4, SHV-2, CTX-M-9 and CTX-M-10) and emergence of new ESBLs (TEM-110, SHV-11, SHV-12, CTX-M-14 and CTX-M-15). A polyclonal structure, including epidemic clones with specific ESBLs (TEM-4, SHV-12 and CTX-M-15), was observed. Phylogenetic analysis showed that most isolates (74.6%) belonged to KpI-type with a clear relationship between KpIII-type and CTX-M-10 producers. Persistence of specific plasmids associated with specific ESBLs (TEM-4, SHV-12, CTX-M-10 and CTX-M-15) was observed. Co-resistance analysis revealed an increment in resistance to trimethoprim (41.5% versus 10.3%), sulphonamide (54.7% versus 29.3%) and nalidixic acid (34.0% versus 6.9%) when compared with 1989-2000.K. pneumoniae is still an important ESBL producer in our institution with a complex epidemiology. The main features were a few outbreaks with persistence of specific plasmids, emergence of new enzymes and an increment in community isolates. These results should be taken into account for the implementation of epidemiological containment measures.

This work describes the first electrochemical immunosensor reported for the determination of IL-13 receptor Rα2 (IL-13Rα2), an emerging relevant biomarker in metastatic colon cancer. The approach involves the formation of sandwich immunocomplexes using specific capture (CAb) and biotinylated detector antibodies (BDAb) further labeled with an streptavidin-horseradish peroxidase (Strep-HRP) polymer, onto carboxylic acid-modified magnetic microbeads (HOOC-MBs). Amperometric detection at disposable carbon screen-printed electrodes (SPCEs) using the (H2O2)/hydroquinone (HQ) system was employed to monitor the affinity reactions. The developed immunosensor exhibits a linear calibration plot over the 3.9-100 ng mL-1 concentration range, a LOD of 1.2 ng mL-1 and excellent selectivity against other non-target proteins. The amperometric immunosensor was applied successfully to quantify for the first time the IL-13Rα2 expression in raw lysates of colon cancer cells and to discriminate the metastatic potential of intact cells through recognition of this target extracellular receptor. In comparison with the commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit involving the same immunoreagents, the immunosensor provides a similar LOD in a half-time for the assay.

Five hundred fecal samples from 462 patients (68.4% ambulatory) (February–April, 2007) from Madrid (Spain) were screened for extended-spectrum β-lactamase (ESBL) producers using ceftazidime and cefotaxime (1 mg/L) MacConkey (MAC) agar plates and a chromogenic media (chromID ESBL; bioMérieux, Marcy-l'Etoile, France). blaESBL, qnr, aac(6′)Ib-cr, and 16S rRNA methylase genes were assessed. A prevalence of 8.2% of ESBL fecal carriers was observed (8.9% hospitalized, 7.9% nonhospitalized patients), higher than that previously observed (1991, 0.6%; 2003, 7.0%). Sensitivity, specificity, and positive and negative predicted values were 100%, 94.8%, 63%, and 100% for chromID ESBL and 87.8%, 89.8%, 43.4%, and 98.9% for MAC, respectively. ESBL distribution was as follows: CTX-M-9-group, 40% (mainly CTX-M-14); CTX-M-1-group, 26.6% (mainly CTX-M-15); SHV-type, 29% (mainly SHV-12); and TEM-type, 4.4%. These enzymes were found in pulsed-field gel electrophoresis nonclonally related Escherichia coli and Klebsiella pneumoniae isolates. Transferable quinolone resistance was confirmed in CTX-M-9 (qnrS1), CTX-M-15 [aac(6′)Ib-cr, qnrS1], and SHV-12 (qnrB7, qnrS1) producers but not 16S rRNA methylase genes. The chromID ESBL medium was reliable to screen ESBL fecal carriers with a general decrease in the laboratory workload. Time-to-time monitoring of ESBL fecal carriers is useful to ascertain current trend of ESBL epidemiology.

Prevalence of extended-spectrum β-lactamases (ESBL) and/or carbapenemase-producing Enterobacteriaceae (EPE and CPE) in stool samples from 75 travellers, 8 people visiting friends and relatives and 3 immigrants who had travelled or came from tropical or subtropical areas was determined. Thirty-one per cent (27/86) of the subjects were faecal carriers of EPE, and 37 EPE isolates were recovered (36 Escherichia coli, 1 Klebsiella pneumoniae). CTX-M-15 was the most prevalent enzyme (64.8%) mainly associated with E. coli belonging to phylogroup A and sequence type complex 10. Most of the ESBL-positive travellers (50%) had visited countries from Asia.

This paper describes a dual immunosensor using neutravidin-functionalized magnetic microbeads (Neu-MBs) and dual screen-printed carbon electrodes (SPdCEs) for the simultaneous amperometric determination of two emerging biomarkers related to breast cancer (BC) and metastasis: Receptor Activator of Nuclear Factor-κB Ligand (RANKL) and Tumor Necrosis Factor alpha (TNF). In the implemented methodology, sandwich-type immunocomplexes, using biotinylated specific capture, detector antibodies and HRP-labeled secondary antibodies, are formed onto Neu-MBs. Electrochemical detection was performed by amperometry (−0.20 V vs. the Ag pseudo-reference) electrode using the H2O2/hydroquinone (HQ) system upon capturing the Neu-MBs modified with the sandwich immunocomplexes for each target biomarker on the corresponding working electrode (WE) of SPdCEs. The approach exhibits high sensitivity offering detection limits of 2.6 and 3.0 pg mL−1 for RANKL and TNF, respectively, using simple protocols and taking 90 min as assay time. The usefulness of the dual immunoplatform was tested by determining RANKL and TNF levels in 5 μL of human serum from healthy controls and BC patients diagnosed with different HER2 subtypes. Results showed a higher expression of both biomarkers in BC patients (38 and 17 % higher for RANKL and TNF, respectively) and were in agreement to those obtained using the ELISA methodologies for each target biomarker involving the same immunoreagents. The obtained results show the potential of this immunoplatform to improve the reliability of BC diagnosis using fast and cost-effective procedures.

To compare the capability of biofilm development between Streptococcus pneumoniae isolates from cystic fibrosis (CF) respiratory samples and those from non-CF blood cultures. Antibiotic susceptibility of biofilm-forming isolates, as well as differences between antibiotic susceptibility of sessile cells [minimum biofilm inhibitory concentration (MBIC)] and their planktonic counterparts (conventional MIC), were also assessed.Biofilm formation was performed using a microtitre method in 20 CF and 22 non-CF blood culture S. pneumoniae isolates.Biofilm formation occurs more frequently among S. pneumoniae isolates from CF (80%) than among non-CF blood culture isolates (50%) (P = 0.04). Moreover MBICs were significantly higher than conventional planktonic MICs among CF but not among non-CF blood isolates, suggesting a high adaptability of CF strains to form biofilms in adverse conditions.

Matrix metalloproteinase 9 (MMP-9) is a zinc-dependent endopeptidase that promotes angiogenesis, tumor growth, metastasis and cell invasion through the degradation of extracellular matrix. This work reports a magnetic microbeads (MBs)-based sandwich immunoassay for the amperometric determination of MMP-9 at screen-printed carbon electrodes (SPCEs). The suitable capture antibody (cAb) is immobilized onto carboxylic MBs to selectively capture the antigen which is sandwiched with a biotinylated detector antibody (biotin-dAb) further conjugated with a commercial streptavidin-horseradish peroxidase (Strep-HRP) polymer. This immunoplatform provides great analytical characteristics in terms of selectivity and sensitivity, achieving a LOD value of 2.4 pg mL-1 for standards in buffered solutions. Although this value is similar to those reported for some other approaches described so far, the method described here is simpler involving a single 30 min incubation step which makes it ideal for automation or implementation in POC devices. Moreover, the method was assayed for the accurate determination of endogenous MMP-9 in both cancer cell lysates and serum samples of patients diagnosed with different subtypes of breast cancer (BC) after a simple dilution. The results obtained show that the disposable and affordable immunoplatform developed is able not only to discriminate BC patients from healthy individuals but also to do it for the worst outcome triple negative (TNBC) subtype.

In this study, an integrated characterisation through polyphenol and caffeine content and antioxidant activity was combined with chemometric analysis to assess the effects of simulated in vitro gastrointestinal digestion on the bioaccessibility of these bioactive compounds from nine different tea infusions. Tea infusions were characterised based on total flavonoids, total polyphenols and antioxidant activity, together with the determination of individual polyphenol content. Fourteen phenolic compounds, including phenolic acids, stilbenes and flavonoids, were selected based on their reported bioactivity and high accessibility, attributed to their low molecular weight. Both polyphenols and caffeine were initially monitored in raw tea infusions and through the different digestion stages (salivary, gastric and duodenal) by capillary high performance liquid chromatography coupled to diode array detection (cHPLC-DAD) and/or HPLC coupled to a triple quadrupole mass analyser (HPLC-MS/MS). Multivariate analysis of the studied bioactives, using principal component analysis and cluster analysis, revealed that the decaffeination process seems to increase the stability and concentration of the compounds evaluated during digestion. The greatest transformations occurred mainly in the gastric and duodenal stages, where low bioactivity indices (IVBA) were shown for resveratrol and caffeic acid (IVBA = 0%). In contrast, the polyphenols gallic acid, chlorogenic acid and quercetin gave rise to their availability in white, green and oolong infusion teas (IVBA > 90%). Furthermore, highly fermented black and pu-erh varieties could be designated as less bioaccessible environments in the duodenum with respect to the tested compounds.

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS2/MWCNTs nanostructures and using horseradish peroxidase (HRP) were implemented. Amperometric detection was carried out at screen-printed dual carbon electrodes (SPdCEs) through the hydroquinone HQ/H2O2 system. The developed dual immunosensor provided limit of detection (LOD) of 0.08 and 0.06 ng mL-1 for BAFF and APRIL, respectively, and proved to be useful for the determination of both cytokines in cancer cell lysates and serum samples from patients diagnosed with autoimmune diseases and cancer. The obtained results agreed with those found using ELISA methodologies.

DNA point mutation in a BRAF proto-oncogene, V600E, is considered an important prognostic and predictive biomarker in various types of cancer, such as melanoma or colorectal cancer. We report here a novel electrochemical (EC) bioplatform for the analysis of BRAF V600E mutation coupled with rolling circle amplification (RCA) and locked nucleic acid (LNA) capture probes. A dual detection system was implemented, whereby two padlock probes complementary to either wild-type (wt) BRAF gene or DNA with V600E mutation (mut) led to amplification of wt or mut variant, respectively. Hybridization with specific LNA capture probes then increased the assay specificity, while EC detection provided rapid measurement times. The bioplatform was applied to analyze BRAF V600E mutation of cancer cells and tumor tissues from patients with melanoma or colorectal cancer. This is the first RCA-based EC bioplatform for BRAF analysis in a dual format without using PCR or sophisticated instrumentation.

ABSTRACT Punctual mutations in the TEM-1 or TEM-2 gene may lead to inhibitor-resistant-TEM (IRT) β-lactamases with resistance to β-lactam-β-lactamase inhibitor combinations and susceptibility to cephalosporins. The aim of this work was to analyze the current epidemiology of IRT β-lactamases in contemporary clinical Escherichia coli . Isolates were prospectively collected in our hospital (2007 and 2008) from both outpatients (59.8%) and hospitalized patients (40.2%). The genetic relationships of the isolates were determined by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and phylogenetic group analysis. IRT genes were sequenced and located by hybridization, and the incompatibility group of the plasmids was determined. From a total of 3,556 E. coli isolates recovered during the study period, 152 (4.3%) showed reduced susceptibility to amoxicillin-clavulanate, with 18 of them producing IRT enzymes (0.5%). These were mostly recovered from urine (77.8%). A high degree of IRT diversity was detected (TEM-30, -32, -33, -34, -36, -37, -40, and -54), and the isolates were clonally unrelated but were mostly associated with phylogenetic group B2 (55.5%). In 12 out of 16 (75%) isolates, the bla IRT gene was plasmid located and transferred by conjugation in 9 of them, whereas chromosomal localization was demonstrated in 4 isolates (25%). The sizes of the plasmids ranged from 40 kb (IncN) to 100 kb (IncFII, IncFI/FIIA), and they showed different restriction patterns by restriction fragment length polymorphism analysis. Unlike extended-spectrum β-lactamase producers, the frequency of IRT producers remains low in both community and hospital settings, with most of them causing urinary tract infections. Although bla IRT genes are mainly associated with plasmids, they can be also located in the chromosome. Despite this situation, clonal expansion and/or gene dispersion was not observed, denoting the independent emergence of these enzymes.

ABSTRACT The chromogenic βLacta test developed for the rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum β-lactamase (ESBL) producers (97.5% true-positive results). However, false-negative results occurred with chromosomal AmpC hyperproducers and plasmid AmpC producers, whereas uninterpretable results were mostly due to VIM-1 carbapenemase producers and possibly low levels of expressed ESBLs.

This paper reports the development of an amperometric immunosensing platform for the determination of cadherin-17 (CDH-17), an atypical adhesion protein involved in the progression, metastatic potential, and survival of high prevalence gastric, hepatocellular, and colorectal tumors. The methodology developed relies on the efficient capture and enzymatic labeling of the target protein on the magnetic microparticles (MBs) surface using commercial antibodies and amperometric transduction at screen-printed carbon electrodes (SCPEs) through the HRP/H2O2/HQ system. The developed immunosensing platform allows the selective determination of the target protein at low ng mL–1 level (LOD of 1.43 ng mL–1) in 45 min and using a single incubation step. The electrochemical immunosensor was successfully used for the accurate determination of the target protein in a small amount (0.5 μg) of raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade.

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producing Enterobacteriaceae from hospital (n = 102) and environmental (n = 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

Alzheimer's disease (AD) is a common neurodegenerative disorder associated with massive degradation of neuronal cell structure due to aging. In recent years, neurofilament light chain (NfL) arises as one of the most promising blood biomarkers for AD. Bioassays available for NfL determination in blood (ELISA kits or Simoa assays) require multiple steps, specialized personnel and expensive instrumentation of application limited to centralized and high-resource environments. With the aim of overcoming these drawbacks, this work reports the first magnetic microbeads (MBs)-based electrochemical bioplatform developed so far for the determination of NfL. The developed platform relies in a sandwich type immunoassay involving an HRP-secondary antibody to enzymatically label the detector antibody on the surface of commercial MBs functionalized with carboxylic acids. Amperometric transduction is carried out at screen-printed carbon electrodes (SPCEs) upon deposition of the magnetic bioconjugates on the working electrode surface using the H2O2/hydroquinone (HQ) system. This MBs-based immunoplatform provides a LOD value of 3.0 pg mL−1. The analytical performance is appropriate for the determination of NfL in plasma and brain tissues from AD patients using simple and short protocols (60 min) and requiring low amount of samples (5 µL raw plasma and 0.1 µg tissue extracts).

This work reports the first dual magnetic beads (MBs) assisted immunoplatform for the simultaneous determination of BAFF (B cell activation factor) and APRIL (a proliferation-induced ligand), two cytokines related to immunity and tumour invasion, growth and metastasis. The electrochemical immunoplatform involves sandwich-type immunoassays implemented on magnetic microparticles functionalized with neutravidin (NA-MBs) or carboxylic groups (HOOC-MBs), and amperometric detection (Eapp = - 0.20 V vs. Ag pseudo-reference electrode) at screen-printed dual carbon electrodes (SPdCE) using the H2O2/hydroquinone (HQ) system. The developed immunosensors provide improved sensitivity, with LOD values of 0.33 and 16.4 pg mL-1 for BAFF and APRIL, respectively, and much shorter assay time that those claimed for ELISA kits and allow their simultaneous determination. The dual immunosensor permits discrimination between healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) or colorectal cancer (CRC) through the determination of both cytokines in 100-times diluted human sera with results in agreement with those provided by the individual ELISA methodologies.

ABSTRACT An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and bla CTX-M-2 was characterized from Escherichia coli . The integron contained an unusual gene cassette array, estX-aadA1 , embedded between the 5′-conserved segment (5′-CS) and 3′-CS1 regions and was flanked by mer -Tn 21 sequences downstream of the tni truncated module. This element constitutes one of the few examples of CR1-bearing class 1 integrons that has been fully characterized.

We present results of microbiological analysis of the first Enterobacterales isolates that were isolated in 2012 in our institution expressing OXA-48 carbapenemase. This enzyme confers resistance to carbapenems, an important group of antibiotics widely used in the hospitals. OXA-48 carbapenemase is currently present in many parts of the world, but it is found particularly frequently in the Mediterranean area. It was disseminated at the Ramón y Cajal Hospital and found to be associated with a particular Klebsiella pneumoniae strain, so-called high-risk clone ST11, which was previously found in our institution in association with other enzymes such as CTX-M-15, VIM-1, and KPC-3. This clone might have acquired a plasmid harboring the bla OXA-48 gene. Our results point out the importance of local epidemiology in the dissemination and maintenance of multidrug-resistant bacteria.

Abstract An electrochemical biosensing platform for serum autoantibodies (AAbs) detection is reported in this work, exploiting for the first time six Alzheimer's disease (AD)‐specific phage‐derived and frameshift aberrant HaloTag peptides as receptors, immobilized on magnetic microbeads (MBs) surface and captured on disposable electrodes to perform amperometric detection. Operational analytical characteristics and clinical diagnostic ability of the bioplatform were probed in optimized key experimental conditions by analysing serum AAbs of AD patients and healthy subjects. The value of 100 % obtained for AUC, sensitivity, and selectivity from the all peptides combined ROC curve, indicate full AD‐diagnostic capability of the methodology, which was further implemented, as proof of concept, in a POC multiplexing platform to detect the signature in a single test over clinically actionable times (1 h 15 min), opening great promise for the type of diagnosis and AD patients’ monitoring follow‐up currently pursued.

This work reports the first electroanalytical bioplatform to date for the determination of antibodies against aquaporin-4 (AQP4-Abs), whose serum level is considered as relevant biomarker for certain autoimmune diseases. The bioplatform relies on the use of magnetic microparticles modified with the biotinylated protein for the capture of specific antibodies. The captured IgGs are enzymatically labelled with a secondary antibody conjugated to the horseradish peroxidase (HRP) enzyme. Amperometric transduction is performed using the H2O2/hydroquinone (HQ) system, which results in a cathodic current variation directly proportional to the concentration of the target antibodies. The evaluation of the analytical and operational characteristics of the developed bioplatform shows that it is competitive in terms of sensitivity with the only biosensor reported to date as well as with the commercially available ELISA kits. The achieved limit of detection value is 8.8 pg mL-1. In addition, compared to ELISA kits, the developed bioplatform is advantageous in terms of cost and point of care operation ability. The bioplatform was applied to the analysis of control serum samples with known AQP4-Abs contents as well as of sera from healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) and Alzheimer (AD) diseases, providing results in agreement with the ELISA methodology.

Development and comparison of the first electrochemical bioplatforms for determining anti-centromere B antibodies (CENPB-Abs) developed in magnetic microbead-assisted or integrated formats using His-tag chemistry.

Currently, there is a need for fast and sensitive analytical methods for monitoring metals in water due to the progressive increase in the presence of metal ions in the environment. These metals reach the environment mainly from industrial activity and heavy metals are non-biodegradable. The present work evaluates different polymeric nanocomposites to carry out the simultaneous electrochemical determination of Cu, Cd, and Zn in water samples. Screen-printed carbon electrodes (SPCE) were modified with the nanocomposites, which were obtained by a mixture of graphene, graphite oxide, and polymers, such as polyethyleneimide, gelatin, and chitosan. These polymers have amino groups in their matrix, giving the nanocomposite the ability to retain divalent cations. However, the availability of these groups plays a fundamental role in the retention of these metals. The modified SPCEs were characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. The electrode that presented the best performance was selected to determine the concentration of metal ions in water samples by square-wave anodic stripping voltammetry. The obtained detection limits were 0.23 μg L-1, 0.53 μg L-1, and 1.52 μg L-1 for Zn(II), Cd(II), and Cu(II), respectively, with a lineal range of 0.1-50 μg L-1. The obtained results made it possible to conclude that the method developed using the SPCE modified with the polymeric nanocomposite presented adequate LODs, reasonable sensitivity, selectivity, and reproducibility. Besides, this platform is an excellent tool for developing devices to simultaneously determine heavy metals in environmental samples.

Alzheimer's disease (AD), in addition to being the most common cause of dementia, is very difficult to diagnose, with the 42-amino acid form of Aβ (Aβ-42) being one of the main biomarkers used for this purpose. Despite the enormous efforts made in recent years, the technologies available to determine Aβ-42 in human samples require sophisticated instrumentation, present high complexity, are sample and time-consuming, and are costly, highlighting the urgent need not only to develop new tools to overcome these limitations but to provide an early detection and treatment window for AD, which is a top-challenge. In recent years, micromotor (MM) technology has proven to add a new dimension to clinical biosensing, enabling ultrasensitive detections in short times and microscale environments. To this end, here an electrochemical immunoassay based on polypyrrole (PPy)/nickel (Ni)/platinum nanoparticles (PtNPs) MM is proposed in a pioneering manner for the determination of Aβ-42 in left prefrontal cortex brain tissue, cerebrospinal fluid, and plasma samples from patients with AD. MM combines the high binding capacity of their immunorecognition external layer with self-propulsion through the catalytic generation of oxygen bubbles in the internal layer due to decomposition of hydrogen peroxide as fuel, allowing rapid bio-detection (15 min) of Aβ-42 with excellent selectivity and sensitivity (LOD = 0.06 ng/mL). The application of this disruptive technology to the analysis of just 25 μL of the three types of clinical samples provides values concordant with the clinical values reported, thus confirming the potential of the MM approach to assist in the reliable, simple, fast, and affordable diagnosis of AD by determining Aβ-42.

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real-time PCR assays for the direct detection and quantification of nine virulence-associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype-related genes (wzxO104 , fliCH4 , rbfO157 , fliCH7 ) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbfO157 /fliCH7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzxO104 /fliCH4 ) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzxO104 /fliCH4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly-virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock.

Performance of the CHROMagar mSuperCARBA media was assessed in both well-characterized carbapenem-resistant Enterobacteriaceae (n = 52) and routine surveillance rectal swab specimens (n = 211). Limit of detection ranged between 101 and 102 CFU/mL except for OXA-48 producers with low-carbapenem MICs (106 CFU/mL). High sensitivity (100%) and specificity (100%) were obtained with rectal swabs.

This work summarized the results obtained in an institutional Klebsiella pneumoniae surveillance program recently implemented in Cuba. Eighteen hospitals from five regions provided a total of 228 K. pneumoniae isolates (164 from admitted patients, four from hospital environmental sources, and 60 isolates from community patients). The genetic relationship was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. Antimicrobial susceptibility was determined by the agar dilution method, and bla(ESBL) genes were sequenced. Fifty four K. pneumoniae isolates were extended-spectrum β-lactamases (ESBL)-producers (23.6%), mostly due to the CTX-M-15 enzyme (79.6%). ESBL isolates were grouped in 27 different sequence types (STs), being the most prevalent ST15 (15%), ST152 (13%), and both ST48 and ST147 (11%, respectively). Community-acquired criteria could be demonstrated in 60 patients (26%) corresponding to urological (33%), wound (27%), respiratory (27%), and otic (13%) infections. Population structure analysis showed that our isolates corresponded to a highly polyclonal population with 10 nonpreviously described STs, demonstrating the importance of local epidemiological studies. We report the first data of the population structure of ESBL-producing K. pneumoniae isolates obtained in a national multicenter surveillance Cuban program. Results showed that a highly polyclonal ESBL-producer K. pneumoniae population was mainly due to CTX-M-15 carriage, whereas carbapenemases production was not present.

Abstract This work reports the development of the first integrated electrochemical immunosensor for the determination of the ligand receptor activator nuclear factor‐κB (RANKL), a biomarker that plays an important role in the regulation of bone resorption process and, as reported recently, in oncology by modulating the immune system response facilitating the tumor growth and metastatic process during cancer development. The developed platform involves a sandwich‐type immunoassay with covalent immobilization of biotinylated capture antibodies onto streptavidin/4‐aminobenzoic acid ( p ‐ABA) grafted screen‐printed carbon electrodes (bCAb‐Strep/ p ‐ABA‐SPCE) and the use of an hybrid nanomaterial composed of gold nanoparticles (AuNPs) and multi‐walled carbon nanotubes (MWCNTs) as nanocarrier of multiple detector antibody and HRP molecules. Amperometric detection with the H 2 O 2 /hydroquinone (HQ) system allowed the construction of a linear calibration plot for RANKL standards between 10.4 and 1,000 pg mL −1 with a LOD of 3.1 pg mL −1 . The determination implied simple handling protocols and short assay times. The developed immunosensor was applied to the determination of RANKL in human serum of patients diagnosed with colorectal cancer (CRC) or rheumatoid arthritis (RA). Each determination required only 5 μL of undiluted serum and took less than 2 h counting since the preparation of blocked‐bCAb‐Strep/ p ‐ABA‐SPCE.

Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus.

We describe the genetic background of bla(TEM-4) and the complete sequence of pRYC11::bla(TEM-4), a mosaic plasmid that is highly similar to pKpQIL-like variants, predominant among TEM-4 producers in a Spanish hospital (1990 to 2004), which belong to Klebsiella pneumoniae and Escherichia coli high-risk clones responsible for the current spread of different antibiotic resistance genes. Predominant populations of plasmids and host adapted clonal lineages seem to have greatly contributed to the spread of resistance to extended-spectrum cephalosporins.

Abstract This paper reports the development of a dual immunosensor using magnetic microcarriers (MBs) and amperometric transduction at dual screen‐printed carbon electrodes (SPdCEs) for the simultaneous determination of two biomarkers: interleukin‐13 receptor α2 (IL‐13Rα2) and E‐cadherin (E‐CDH), with both extracellular and soluble fraction; oncogenic and tumor suppressor markers, respectively, of great relevance in metastatic processes. The implemented methodology involved the formation of sandwich‐type immunocomplexes using specific capture antibodies immobilized onto carboxylic acid magnetic microbeads (HOOC‐MBs), and biotinylated detector antibodies labeled with streptavidin−horseradish peroxidase conjugates (Strep‐HRP). The amperometric detection was performed by addition of hydrogen peroxide in the presence of hydroquinone (HQ) as the redox mediator. The dual immunosensing platform provided linear calibration ranges suitable for the determination of both biomarkers in liquid and solid clinical specimens as well as excellent selectivity against other cancer biomarkers. This simple handling dual bioplatform was applied to the determination of the soluble and extracellular fraction of the target biomarkers in serum and paraffined‐embedded tissues from colorectal cancer (CRC) patients diagnosed at different tumor grade. The obtained results reveal great potential of this configuration to improve the reliability in diagnosing metastatic CRC.

First electrochemical bioplatforms described for the single and simultaneous determination of two immunoglobulin (Ig) subtypes, IgE and IgG4 specific to ovalbumin (OVA), considered as reliable markers for the diagnosis and monitoring of egg allergy.

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum beta-lactamases (ESBLs) were studied. Clinical records of patients with ESBL-positive K. pneumoniae isolates between 1989 and 2003 (n = 80) were reviewed retrospectively. Patients with SHV- and TEM-type ESBLs were identified more frequently in the intensive care units (67% and 78%, respectively), whereas those with CTX-M ESBLs were found in medical wards (52.2%) or were outpatients (17.4%) (p <0.01). The absence of urinary or central catheters was associated with CTX-M-10 (p 0.013 and p <0.01, respectively). Central catheter-related infections and secondary bacteraemia were associated more frequently with SHV- and TEM-type ESBLs, whereas urinary tract infections were associated with CTX-M-10. Previous aminoglycoside use was associated particularly with SHV-type ESBLs (p <0.01), whereas amoxycillin-clavulanate and oral cephalosporins were associated with CTX-M-10 (p <0.01 and p 0.050, respectively). The frequency of adequate empirical treatment was low (22%), and 61% of patients were treated according to the susceptibility testing results. Mortality (22%) and related mortality (14%) did not differ statistically according to the type of ESBL. Different ESBL types in K. pneumoniae were associated with different clinical variables, and this should be taken into account in current and future epidemiological scenarios.

Abstract Alzheimer's disease (AD), the most common neurodegenerative disorder, demands new cost‐effective and easy‐to‐use strategies for its reliable detection, mainly in the preclinical stages. Here, we report the first immunoplatform for the electrochemical multidetermination of four candidate protein biomarkers in blood, neurofilament light chain (NfL), Tau, phosphorylated Tau (p‐Tau) and TAR DNA‐Binding Protein 43 (TDP‐43). It involves implementation of sandwich‐type immunoassays and enzymatic labelling with horseradish peroxidase (HRP) on the surface of magnetic microbeads (MBs). Amperometric detection is performed after depositing the magnetic immunoconjugates on disposable quadruple transduction platforms by monitoring the enzymatic reduction of H 2 O 2 mediated by hydroquinone (HQ). The immunoplatform achieved LOD values smaller than the content of target biomarkers in plasma of healthy subjects, with RSD values&lt;5 %, and lower cost and shorter assay time (60–90 min) than other available methodologies and was applied to the analysis of plasma from healthy controls and AD patients.

a7PAMspreserve spatio-temporal patternsofnAChRactivationand unlike AChE inhibitors, activate only the a7 nAChR subtype, which should provide a better side effect profile. We have performed detailed characterization of BNC375, a Type I positive allosteric modulatorofa7nAChR.ActionofBNC375ona7nAChRs invitrowas examined in GH4C1 cells stably, expressing rat a7 nAChR. Manual patch-clamp recordings were made with a fast-application add-on (Dynaflow Resolve, Cellectricon, Sweden). A concentration– response curve of a7 nAChR potentiation by BNC375 yielded an EC50 1.9mM, nH 1.62, Emax 1572% for peak current and 1.3mM, 1.60, 2616% for areaunder curve (AUC), respectively. In the presence of 2mM ( EC50) BNC375 the concentration–response of acetylcholine exhibited 10 fold leftward shift (EC50 14.5mM vs. 127.7mM, nH 1.66 vs. 1.76) as well as 246% potentiation of a saturating ACh concentration. BNC375 had a minimal effect on the kinetics of potentiated currents and in contrast to a classical Type II a7 PAM PNU-120596, was not able to re-activate a7 nAChRs after desensitization with 0.5–1 mM ACh. The cognition-enhancing properties of BNC375 were evaluated using reversal of scopolamine-induced memory deficits in the mouse T-maze Continuous Alternation Task (T-CAT) and the rat novel object recognitionmodel (NOR). In the T-CAT, BNC375 at doses from 0.003 to 3 mg/kg p.o. dose-dependently reversed the memory-impairing effect of scopolamine from0.03 mg/kg. In the NOR all three doses (0.1, 0.3, 1 mg/kg p.o.) showed a significant reversal of scopolamine-induced deficits. In summary, we show that BNC375 potently enhances a7 nAChRmediated currents in vitro and has a favourable PAM Type I-like kinetic profile. The compound is efficacious in a broad range of agonist concentrations – from subthreshold to saturating. BNC375 demonstrates cognition-enhancing properties in two different in vivo animal models of episodic and working memory with a broad therapeutic window (100 fold).

Introduction: One of the mechanisms governing protein denitrosylation is the system thioredoxin/thioredoxin reductase (Trx/TrxR).Alteration of this enzymatic system may impair S-nitrosothiol (SNO) homeostasis in tumor cells, providing new insights into the role of nitric oxide in cancer and its role in tumor progression and antitumoral treatment.Material and Method: MCF-7, MDA-MB-231 and BT-474 cells were pretreated or not with the highly specific TrxR inhibitor auranofin and exposed to different doses of S-nitroso-L-Cysteine (CSNO).S-nitrosylated proteins were detected using the 'biotin-switch' assay.Subcelullar localization of ER-alpha was analyzed by confocal microscopy.Oncomine database was explored for TrxR (TXNRD1) expression in ER-and ER+ breast tumors.Results: In all the three cell lines, a high CSNO dose (500 mM) reduced cell proliferation and this effect was potentiated by pretreatment with auranofin.Augmented levels of S-nitrosylated proteins were observed in MCF-7 cells treated with 500 mM CSNO, and this effect also was potentiated by pretreatment with auranofin.However, treatment with auranofin and 100 nM CSNO enhanced cell proliferation of estrogen receptor positive (ER+) MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53), or BT-474 (ER+, mut p53) cells.This cell growth was associated with Akt and Erk 1/2 phosphorylation, augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-a.The specific silencing of ER-alpha expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition.Estrogenic deprivation potentiated the pro-proliferative effect of SNO homeostasis impairment in MCF-7 cells.Moreover, the subcelullar distribution of ER-alpha was altered, with a predominantly nuclear localization in those cells with impaired SNO homeostasis.When Oncomine database was interrogated, positive ER status in breast tumors was found to be associated with significantly lower levels of TXNDR1 expression.Conclusion: ER status in breast cancer may dictate the tumor response to different nitrosative environments.Factors impairing the Trx/TrxR enzymatic system in tumor cells may confer survival advantages to ER+/p53 wt breast tumors.These molecular mechanisms may also play a significant role in the development of resistance against hormonal therapies that arise in this type of mammary tumors.

Abstract Background: Much is known about the mechanisms involved in the response to anti-hormonal treatment or those involved in the response to antiangiogenic therapy. It is also known the association between angiogenesis and hormonal status, both in physiological and pathological settings. However, the molecular and cellular mechanisms contributing to the efficacy of combined antiangiogenic-antihormonal therapy in breast cancer are still unknown. This combination is currently in clinical trials, but unfortunately there are scarce preclinical studies contributing to the rationale of combining antiangiogenic and antihormonal therapies. Aims: To define the mechanisms involved in the response to combined antiangiogenic-antihormonal treatments in breast cancer cells. Methods: Breast cancer cell lines with different estrogen dependence (MCF-7, BT-474, MDA-MB-231) were subjected to an estrogen gradient (estradiol), and treated with fulvestrant (antiestrogen), plus bevacizumab (anti-angiogenic). Cellular proliferation and apoptosis were determined using the corresponding kits. Proliferation and survival intracellular signaling pathways, estrogen alpha and VEGF receptors activation and COX-2 expression were analyzed by western-blot using specific antibodies. Results: In estrogen-dependent breast cancer cells (MCF-7 and BT-474) the pro-proliferative effect of estradiol decreased after bevacizumab treatment. Furthermore, the combination of the antiangiogenic with an antiestrogen enhanced this antiproliferative effect, that was also related to the reduction in the levels of VEGF-A in the culture medium and to diminished ER alpha phosphorylation. The combined treatment also altered the phosphorylation of Akt and Erk1/2 signaling kinases. Interestingly, bevacizumab treatment augmented COX-2 activity in MCF-7. Conclusions: Our results suggest that in estrogen dependent breast cancer cells the anti-proliferative effect of bevacizumab depends on estradiol concentration, that in turn affects VEGF production levels, using a different mechanism to apoptosis. The combination of bevacizumab with antiestrogens enhances this antitumoral effect, altering intracellular signaling pathways of proliferation and survival. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-07.

The Cover Feature illustrates a quadruple immunoplatform for the amperometric determination of blood protein candidate biomarkers (NfL, Tau, p-Tau and TDP-43) to assist in the reliable and minimally invasive Alzheimer′s disease diagnosis. The cover was drawn by Beatriz Arévalo and co-designed with the authors. More information can be found in the Research Article by A. Valverde, J. M. Gordón Pidal et al.

In this paper, a novel AI method for failure prediction in transmission lines components is presented. The method combines machine learning and deep learning capabilities. The approach was tested using degradation simulated data of a composite insulator exposed to different levels of environmental pollution. The failure model was constructed using historical real data of a Mexican utility. Preliminary experimental results shows that the joint use of deterministic forecasting and probabilistic diagnosis methods help determine the future failure of a transmission line component for different time horizons with very acceptable precision rates.

El pseudotumor cerebri (PC) es un síndrome caracterizado por hipertensión intracraneal en ausencia de lesión ocupante de espacio, hidrocefalia, trombosis de senos venosos cerebrales y de anomalías bioquímicas o citológicas del líquido cefalorraquídeo. Se ha asociado a diversos factores como enfermedades sistémicas o fármacos. Presentamos una paciente, diagnosticada de lupus eritematoso sistémico (LES) 7 años antes, con un cuadro agudo de cefalea, vómitos y borrosidad visual acompañado de papiledema bilateral secundario a un PC. Se inició tratamiento con altas dosis de esteroides, con rápida resolución de los síntomas y del papiledema. Pseudotumor cerebri (PC) is a syndrome characterized by intracranial hypertension in the absence of any space-occupying lesion, hydrocephalus, cerebral sinus thrombosis and biochemical or cytological abnormalities in the CSF. PC has ben associated with several factors such as systemic conditions or drugs. We report here the case of a patient who presented with headache, vomiting and blurred vision accompanied by bilateral papilledema and had been diagnosed with systemic lupus erythematosus (SLE) seven years before. Treatment was started with high-dose corticosteroids with rapid resolution of the clinical symptoms and papilledema of the patient.

The cover feature illustrates an electrochemical bioplatform with full diagnostic capability for Alzheimer's disease exploiting for the first time the use of six phage-derived and frameshift aberrant HaloTag peptides as receptors immobilized on magnetic microbeads, which were captured on multiplexed disposable electrodes to perform amperometry detection of specific serum autoantibodies. Cover design by Eloy Povedano and Víctor Ruiz Valdepeñas-Montiel. More information can be found in the Communication by Dr. R. Barderas, S. Campuzano, J. M. Pingarrón and co-workers.

•El cáncer colorrectal es una de las neoplasias que cumplen criterios para realizar un cribado: importancia de la enfermedad, historia natural y tratamiento efectivo en fases iniciales.•Los métodos de cribado más estudiados son la sangre oculta en heces, la sigmoidoscopia y la colonoscopia.•Los beneficios del cribado se centran en la reducción de la incidencia y la mortalidad.•Los efectos adversos del cribado más importantes son los falsos positivos y los falsos negativos.•El balance beneficios y efectos adversos debe tenerse en cuenta al aconsejar el cribado.

As an increase in anaerobes resistant to a great variety of antimicrobials have been seen in the last few years, a search for new agents with activity against these microorganisms is needed. One of these agents is trovafloxacin. In our study, all strains were susceptible to imipenem, metronidazole, chloramphenicol and piperacillin/tazobactam. A total of 97% of the strains were inhibited with 2 mg/ml of trovafloxacin. The microorganisms Bacteroides fragilis and B. uniformis showed the least susceptibility against the antimicrobials studied, with a MIC90 for trovafloxacin of 1 and 2 mg/ml, respectively. Fusobacterium spp. were the most susceptible, with an MIC90 for trovafloxacin of 0.5 mg/ml. Trovafloxacin showed good activity against Gram-negative anaerobe rods, and could therefore be considered as an alternative in the treatment of the infections produced by these microorganisms.